JPS58209990A - Conversion of adenosine monophosphate to adenosine triphosphate - Google Patents

Abstract

PURPOSE:To convert adenosine monophosphate (AMP) essentially completely to adenosine triphosphate (ATP), stably for a long period, by using an immobilized enzyme and adjusting the mixing ratio of AMP to ATP to a specific state. CONSTITUTION:An enzyme capable of converting AMP to adenosine diphosphate (ADP) such as adenylate kinase and an enzyme capable of converting ADP to ATP such as acetate kinase are prepared by microorganisms having an optimum proliferation temperature of 50-85 deg.C. Both enzymes are immobilized separately, and the immobilized enzymes are used in combination for the conversion of AMP via ADP to ATP. In the above conversion process, the concentration of ATP is adjusted to a level satisfying the formula [AMP in the formula is concentration (mM) of AMP, ATP is concentration (mM) of ATP, gamma is the ratio of the activity of the enzyme converting ADP to ATP to that of the enzyme converting AMP to ADP, and is >=1].

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for converting adenosine-phosphate (hereinafter referred to as AMP) to adenosine diphosphate (hereinafter referred to as ATP).

In living organisms, numerous biosynthetic reactions are carried out using enzymes as catalysts to maintain life.

Among them, A
TP is required as an energy source or cofactor. At this time, after ATP functions as an energy source or a cofactor, it is decomposed into adenosine diphosphate (hereinafter referred to as ADP) or AMP and is consumed. On the other hand, recently, many attempts have been made to industrially reproduce in-vivo reactions in reactors in factories. As a result of a review of the modern chemical industry, attention has been paid to the energy-saving and non-polluting properties of biological reactions, and this technology is becoming essential in the field of fine chemicals. It has already been successfully put into practical use in technical fields such as chemical reactions. However, in the binding reaction, as mentioned above, the problem that it is not economically viable because the expensive substance IcATP is consumed as an energy source or a cofactor has hindered its practical application. That is, an important technique for overcoming this problem is to regenerate and convert ADP and AMP, which are products of ATP consumption, especially AMP, which has been consumed at the lowest energy level, into ATP.

From this point of view, various studies have been conducted on the regenerative conversion of ATP. For example, ATP is produced in living organisms through reactions such as glycolysis, and attempts to utilize this production are known.

That is, there is a method in which consumed ATP is regenerated and replenished using microbial cells. but.

This method has not reached a practical level due to side reactions and poor conversion efficiency.

On the other hand, attempts have also been made to utilize ATP converting enzymes, although they are not thermostable enzymes. For example, Whitesides et al. converted AMP obtained by enzymatically converting adenosine to ATP using adenylate kinase and acetate kinase in Escherichia coli (Journal of the American Chemical Society, io).

Vol. 1, p. 304, 1978). In addition, Whitesides et al. immobilized both of the above E. coli enzymes with bromic cyanide and attempted continuous conversion of AMP to ATP.
It has been reported that in the absence of a stabilizer, the residual activity was only a few percent or less, and the long-term stability was also extremely poor (Enzyme Engineering, Vol. 2, 21).
7 pages, 1974, edited by E. K. Pai et al., Plenum Press). Moreover.

Even if an immobilized enzyme is used and a stabilizer is added, one reaction takes a long time and the conversion efficiency is too high.

It cannot be used for long-term operation at the chemical industrial level.

As a result of intensive research into converting and regenerating AMP, which is a product decomposed to the lowest energy level, into ATP, the present inventors used a converting enzyme produced by a microorganism whose optimal growth temperature is 50°C to 85°C. Then, for a short time,
We discovered that AMP can be converted to ATP in high yield and stably over a long period of time.

A patent application was previously filed (Japanese Patent Application No. 10337-1983). However, in this method, the concentration ratio of AMP and ATP is reduced to 1.
:1.

A large amount of expensive ATP must be used, and continuous ATPO conversion at the chemical industrial level is economically unfavorable. Moreover, a phenomenon in which the conversion rate to ATP sometimes fluctuated was observed.

In order to improve this point, the present inventors conducted further intensive research and found that by controlling the mixing ratio of AMP and ATP under specific conditions, AMP can be produced stably for a long period of time at low cost and with good operability. The present invention was completed based on the discovery that virtually -F can be converted into ATP almost 100%.

That is, in the present invention, the optimum growth temperature is 50'C to 85'C.
Immobilization of an enzyme that converts adenosine-phosphate to adenosine diphosphate or an enzyme that converts adenosine diphosphate produced by a microorganism at ℃, and converts the obtained adenosine-phosphate to adenosine diphosphate. When converting adenosine-phosphate into adenosine diphosphate using a combination of an enzyme that converts adenosine diphosphate and an immobilized enzyme that converts adenosine diphosphate into adenosine diphosphate, the concentration of adenosine diphosphate is controlled to a condition that satisfies the following formula (a). This is a method for converting adenosine-phosphoric acid to adenosine diphosphoric acid.

(However, AMP is the concentration of adenosine-phosphate (mM).

ATP represents the concentration (mM) of adenosine diphosphate.

γ is the ratio of the immobilized enzyme activity of the enzyme that converts adenosine diphosphate to adenosine diphosphate and the immobilized enzyme activity of the enzyme that converts adenosine-phosphate to adenosine diphosphate, and represents a positive number greater than or equal to 1. ) The present invention converts AMP into ADP
and converting it into .

It consists of converting the ADP produced here into ATP, and is an enzyme that converts AMP to ADP.

For example, adenylate kinase is used, where A M
ATP is used as phosphate donor to l). Next, examples of enzymes that convert ADP to ATP include:
Many kinases such as acetate kinase, carbamate kinase, creatine kinase, 3-phosphoglycerate kinase, pyruvate kinase, and polyphosphate kinase can be used, but the price of the phosphate donor, the conversion activity to ATP, and the availability of the enzyme In view of ease, it is most advantageous to use acetate kinase, and in this case, acetyl phosphate is used as the phosphate donor. In this way, when using adenylate kinase and acetate kinase, ATP and acetyl phosphate are used as phosphate donors for each enzyme, but ATP as a phosphate donor is converted into ATP, which is the final converted product. Since acetyl phosphoric acid can be recycled and used, it is no longer necessary to supply only acetyl phosphoric acid as a phosphoric acid donor. Another advantage of both enzymes is that such systemization allows for efficient design.

As mentioned above, it is possible to convert A and MP into ATP by using two types of converting enzymes, but these enzymes are enzymes produced by microorganisms whose optimal growth temperature is 50°C to 85°C. It is necessary that there be. Such microorganisms include Bacillus stearothermophilus,
Bacillus plebis.

Microorganisms of the genus Bacillus such as Bacillus coagulans, Bacillus thermoproteoliticus, and Bacillus acidocaldarius, microorganisms of the genus Clostridium, and microorganisms of the genus Thermoactinomyces.

Microorganisms of the genus Achromobacter, microorganisms of the genus Streptomyces, microorganisms of the genus Micropolisvora, microorganisms of the genus Thermus such as Thermus aquaticus, Thermus thermophilus, Thermus fujibus, microorganisms of the genus Thermomicrobium, microorganisms of the genus Calburia. microorganisms, etc.

It also includes microorganisms that grow at room temperature into which the genes of these microorganisms have been introduced. Among these microorganisms, Bacillus stearothermophilus is particularly suitable for producing both enzymes, adelate kinase and acetate kinase.

Both enzymes obtained from this microorganism are easy to purify and have high specific activities.

In the present invention, the above-mentioned enzyme is used after being immobilized. For this purpose, the enzyme is transferred to a suitable carrier, such as a polysaccharide derivative such as cellulose, dextran, agarose, etc.
derivatives of polyamino acids or polyamides such as polystyrene, ethylene-maleic acid copolymers, cross-linked polyacrylamide, etc., derivatives of polyamino acids or polyamides such as L-alanine-glutamic acid copolymers, polyaspartic acid, etc., glass, alumina, It may be bonded to, entrapped in, or adsorbed to an inorganic derivative such as hydroxyapatite, and used by filling a reactor such as a column.

In carrying out the present invention, the temperature at which AMP is converted to ATP is preferably set at around room temperature or below 5° C., which is the optimal growth temperature for enzyme-producing microorganisms. still,
The pH when converting AMP to ATP is near neutral, that is, 6.5 to 11, preferably 6.5 to 9.0.
．． More preferably, a range of 7 to 8 is used. As the buffer solution, common ones suitable for these pHs can be used. For example, around 7, phosphate, imidazole salt, tris hydrochloride, collidine salt, barbital hydrochloride, etc. can be mentioned. Furthermore, in order to promote the reaction between adenylate kinase and acetate kinase more effectively, two kinds of divalent metal ions can be used, and as divalent metal ions, for example, magnesium ions and manganese ions are particularly preferred. Recommended.

To convert AMP to ATP in the present invention, 1 For example, AM
P is 0.1 μM to 4 M, preferably 1 μM to 2 M, more preferably 10 μM to 500 mM, and acetyl phosphate is 0.1 μM to 4 M, preferably 1 μM to 4
o.

mM, more preferably 10 μM to 300 nsM, and ATP that satisfies formula (a) are supplied from one end of a packed bed reactor to produce AMP-+ADP-)ATP in the reactor.
It is possible to perform the conversion of At that time, the reaction liquid eluted from the reactor was analyzed using an appropriate analysis system, and the AM
Each concentration of P, ADP, and ATP and the conversion rate to ATP can be determined. At this time, the flow rate varies depending on the size of one reactor, but an appropriate flow rate can be selected, for example, between 0.3 μt/hour and 1×10t/hour. Also.

The device for supplying AMP, ATP, and acetyl phosphoric acid to the reactor is not particularly limited as long as the flow rate can be changed by an external control signal, but for example, a metering pump driven by a pulse motor can be used. (hereinafter referred to as a variable amount liquid feeding device). moreover,
An automatic control valve is provided between each substrate solution tank and a variable amount liquid feeding device, and the flow rate and concentration of each substrate solution can be changed by controlling the opening and closing of each control valve using an external signal.

For example, a solenoid valve can be used as the automatic four-section valve. Although one reactor may of course be used at room temperature, it is preferable to add means for maintaining the temperature. Also 9
As a system for analyzing the reaction liquid from the reactor, AMP,
For example, the method is not particularly limited as long as it detects ADP and ATP.

Preferably, a high performance liquid chromatography device is used.

The present invention provides long-term multistable and economical AM in the chemical industry.
In order to substantially convert P into 100ch ATP, it is necessary to control so that equation (a) is satisfied.

In particular, by adding the ATP concentration to equation (a), o, osx-,
-xγ2 It is preferable to control it to AMP or less. The following method can be used to control so that equation (a) is satisfied. That is, the formula (a) and the data necessary for the calculation are input into the calculation control device in advance, and from these and the analysis data from the analysis system, the conversion rate of AMP to ATP is calculated, and the above-mentioned variable amount liquid delivery device and A signal may be sent from the arithmetic control device to at least one of the automatic control valves to change the flow rate or concentration and control so as to satisfy equation (a). The data required for the calculation at that time is AM
This is the raw material concentration of P and ATP and the ratio of the immobilized enzyme activity of the enzyme that converts azone-cinniphosphate to adenosine tris-phosphate and the immobilized enzyme activity of the enzyme that converts adenosine-phosphate to adenosine diphosphate. The data are the concentrations of AMP, ADP, and ATP in the reaction solutions from 9 reactors. Further, the arithmetic control device is a device having a calculation function and a function of outputting a control signal to an external device, and for example, a microcomputer can be used. Furthermore, immobilized enzyme activity refers to the activity of an immobilized enzyme.1For example, in the case of adenylate kinase, AMP+ATP→2・AD
In the case of acetate kinase, it shows activity in the direction of ADP.
+acetyl) vI) acid → ATP decacetic acid.

To measure this activity, a predetermined amount of immobilized enzyme.

For example, depending on the degree of activity, 5 to 10 μL was added to the solution for measuring activity, and the absorbance change was followed and measured using a spectrophotometer in the same manner as for free enzyme.

One unit of enzyme activity is the amount that produces 1 micromole of ADP per minute for adenylate kinase at 30°C.
For acetate kinase, 1 micromole of ATP per minute
is the amount produced.

Of course, as the ATP used in the present invention, ATP which is the final converted product of the above reaction may be recycled and used. In this case, only acetyl phosphoric acid needs to be supplied as the phosphoric acid donor.

According to the present invention, in converting AMP to ATP,
It can overcome the variation in conversion rate given in the past.

Moreover, it can be efficiently, continuously and economically converted to ATP over a long period of time at a conversion rate of substantially 100%. Moreover, since it is easy to operate, the ATP conversion rate can be maintained stably over a long period of time. Furthermore, in the present invention, AT as a phosphate donor
One advantage is that P may be converted from AMP to ATP in a packed bed reactor. Furthermore, the starting material does not need to be pure AMP, and may be a mixture of AMP with ADP and ATP as long as it is controlled so as to satisfy the formula (a). Also, when used in the chemical industry, This is a very advantageous point.

In addition, it is performed in vivo as mentioned at the beginning. It becomes possible to operate a so-called bioreactor that performs the binding reaction as an external chemical reaction. For example, protein synthesis reactions and pegtide synthesis reactions by amino acid activating enzymes, which are the most important reactions in living organisms, require ATP as an energy source, and the expensive ATP used at that time is consumed and decomposed into AMP. Therefore, when performing this reaction in vitro, it is practically essential to convert and regenerate AMP into ATP. According to the present invention, it is possible to put such a reaction into practical use, and its value is immeasurable in industry.

In addition, when combining the desired synthesis reaction with the ATP regeneration reaction as described above, a system is also required to separate the generated AMP from the desired product, the reaction product of the phosphate donor (for example, acetic acid, etc.) . This is made possible by techniques such as chromatography, and the desired reaction system,
It will be completed as a system for the chemical industry that combines three systems: an ATP regeneration system and an AMP separation system.

The present invention can be extremely usefully applied to the reuse of ATP in this way, but from a different perspective.

It can also be viewed as a method for producing ATP using AMP as a raw material. That is, ATP is an important substance as a medicine and is produced at an industrial level. However, the current fermentation method has problems such as easy production of by-products and poor productivity.

The price of ATP also had to rise.

However, according to the present invention, these problems can be eliminated at once, and highly purified ATP can be supplied with good productivity. The present invention also includes such objects.

Next, the present invention will be explained in more detail with reference to Examples.

Examples 1-4. Comparative Examples 1 to 3 m Characterized CH-Sepharose 4B (manufactured by Pharmacia)
After washing and swelling 5y, 2000 units of acetate kinase obtained from Bacillus stearothermophilus N CAl3O3 strain (optimum production temperature 60°C) was added and reacted to obtain 1000 units of immobilized acetate kinase. This operation was repeated using 250 units of adenylate kinase instead of acetate kinase to obtain 100 units of immobilized adenylate kinase. At this time, the ratio between the identified enzyme activity of acetate kinase and the immobilized enzyme activity of adenylate kinase was 10.

Both of these immobilized enzymes were packed into a glass column with an inner diameter of 1.6 crn and a length of 10 columns. ? 25mM imidazole hydrochloride buffer containing IM magnesium chloride, PH
Each substrate dissolved in 7,5 was fed to the column at a flow rate of 150 d/hr. This column was equipped with a variable liquid feed device, a magnetic valve, and a microcomputer. The temperature inside the column was maintained at 30°C. A in the reaction solution eluted from the column
MP, ADP, and ATP concentrations were determined using a high performance liquid chromatography device. Note that the AMP concentration was fixed at 1.5 mM, and the acetyl phosphate concentration was fixed at 5 mM.

Next, add 0.063 mM AT using a microcomputer.
P (concentration ratio of ATP and AMP is 0.042° Example 1)
.

0.07mMATP (The concentration ratio of ATP and AMP is 0.07mM ATP.
047゜Example 2), 0.13mM ATP (
The concentration ratio of ATP and AMP is 0.087. Example 3)
, 0.19mM ATP (the concentration ratio of ATP and AMP is 0.127°, Example 4), and the conversion rate to ATP was determined by various changes so as to satisfy equation (a).

As a result, only 20 minutes after supplying to the column, no AMP was detected, and 98.5% of ATP,
1.5% ADP was detected.

Separately, for comparison, the ATP concentration was set to 0.03mM ATP (A
The concentration ratio of TP and AMP was 0.02° (Comparative Example 1).

0.024mM ATP (concentration ratio of ATP and AMP is 0.016° Comparative Example 2), 0.014mM AT
P (The concentration ratio of ATP and AMP is 0.009° Comparative Example 3
) was carried out in the same manner as in Examples 1 to 4.

As a result, in Comparative Example 1, A'i'P was 95%.

In Comparative Example 2, 89% of ATP, 8% of ADP, and 3% of AMP were detected, and in Comparative Example 3, 72% of ATP and 2% of ADP were detected.
20 chips and 8 chips of AMP were detected.

Example 5 After starting the reaction under the same conditions as in Example 2, the eluate from the column was collected 20 minutes later at a concentration ratio of ATP and AMP of 0.047.
It was circulated to the column so that

As a result, after using the eluate from 9 reactors instead of ATP, ATP decreased from 98% to 9% over a period of 20 minutes and 5 hours.
It was maintained between 8.5φ.

Example 6 Immobilized acetate kinase 2000 obtained in the same manner as Example 1
unit, 200 units of immobilized adenylate kinase to an inner diameter of 2.
Packed into a glass column with a length of 12 crn using a zero saw,
50mM imidazole-hydrochloric acid buffer containing 25mM magnesium chloride and 0.04% lium azide.

Pfl 7.5 K dissolved 3,0mM AMP,
0.13mM ATP (concentration ratio of ATP and AMP is 0.
043), 10mM acetyl phosphate at 300rn
AMP and ATP were supplied to the column at a flow rate of v hours to maintain the flow rates and concentrations of AMP and ATP so as to satisfy equation (a).

As a result, the conversion rate to #) ATP was maintained between 98.5% and 99.0% for 10 days after the start of the first reaction.

Example 7 After starting the reaction under the same conditions as in Example 6, 30 minutes later, the eluate from the column (containing 98% ATP) was converted into ATP (
7) Instead, KATP and AMP (7) were circulated and supplied to the column so that the concentration ratio was 0.043.

As a result, the conversion rate to ATP remained at 98.2 to 98.7% for 10 days after the start of one reaction.

Agent Yuzo Kodama

Claims (1)

[Scope of Claims] (11. An enzyme that converts adenosine-phosphate into adenosine diphosphate or an enzyme that converts adenosine diphosphate into adenosine diphosphate produced by a microorganism whose optimal growth temperature is 50°C to 85°C is immobilized. , the obtained adenosine-
When converting adenosine-phosphate to adenosine diphosphate using a combination of an immobilized enzyme that converts phosphoric acid to adenosine diphosphate and an immobilized enzyme that converts adenosine diphosphate to adenosine diphosphate, the concentration of adenosine diphosphate is determined as follows ( b) A method for converting adenosine-phosphoric acid to adenosine diphosphoric acid, which is characterized by controlling the conditions to satisfy the formula. (However, AMP is the concentration of adenosine-phosphate (mM)
. ATP represents the concentration of adenosine diphosphate (mM), and γ
is the ratio of the immobilized enzyme activity of the enzyme that converts adenosine diphosphate to adenosine diphosphate and the immobilized enzyme activity of the enzyme that converts adenosine-phosphate to adenosine diphosphate.
Represents a positive number greater than or equal to 1. ) (2) The enzyme that converts adenosine-phosphate to adenosine diphosphate is adenylate kinase. 2. The conversion method according to claim 1, wherein the enzyme that converts adenosine diphosphate to adenosine diphosphate is acetate kinase.